The invention relates to a process for the quantitative and qualitative determination of antigens, antibodies and their complexes by means of a chemiluminescing labelling substance excited to a chemiluminescence by an analytical reagent, as well as to the use of chemiluminescing triphenylmethane dyes for performing such a process.
It is very important to be able to measure antigens, antibodies or their complexes in secretions, excretions and body fluids of both vertebrate and human organisms. In this way, it is possible inter alia to obtain diagnostic information.
It is known to detect a serological reaction by labelling one or more reaction components with a radioactive isotope by conjugating with an enzyme, a fluorescent dye or a chemiluminescent such as luminol or luciferin.
The radioimmunoassay is described in the Journal Clinical Endocrinology 27, 1967, p.973 and loc.cit. 28, 1968, p.343. The important disadvantage of this assay is that it is necessary to use radiation-emitting isotopes, whilst complicated and costly equipment is required for carrying out such an assay.
When labelling a reaction component with an enzyme, the disadvantage is that it is a complicated procedure to carry out this labelling and the reaction product obtained is difficult to store and use. In addition, the enzymes to be used are biologically active substances of an extremely complex nature, this being the cause of the aforementioned difficulties. In addition, the substrates used for detecting the combined enzyme are carcinogenic, which is disadvantageous. The enzyme assay of this type is described in the Bull.World Health Organ 53, 1976.
In the fluorescence method, a reaction product containing antigens and antibodies is identified by fluorescence, accompanied by irradiation with short-wave light. It is disadvantageous in this connection that the excitation light must be separated from the emitted light by costly equipment.
The chemiluminescents hitherto used for chemiluminescence have been difficult to bond to the reactants of a serological reaction and besides this they lose up to 99.3% of their original luminescence after bonding, cf e.g. Nature, Vol.299, 1979, pp.646-647. It is also reported in the Journal of Immunological Methods 21, 1978, pp.178-184 that the chemiluminescent luminol is for this reason unsuitable for routine clinical laboratory tests.